Rapid detection and identification of ‘Candidatus Phytoplasma pini’-related strains in Lithuania based on genomic markers present in 16S rRNA and tuf genes

Authors: VALIUNAS, DEIVIDAS - Nature Research Centre; JOMANTIENE, RASA - Nature Research Centre; IVANAUSKAS, ALGIRDAS - Nature Research Centre; SNEIDERIS, DONATAS - Nature Research Centre; ZIZYTE-EIDETIENE, MARIJA - Nature Research Centre; Shao, Jonathan; Zhao, Yan; Costanzo, Stefano; Davis, Robert

Submitted to: Forest Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/21/2019
Publication Date: 9/5/2019
Citation: Valiunas, D., Jomantiene, R., Ivanauskas, A., Sneideris, D., Zizyte-Eidetiene, M., Shao, J.Y., Zhao, Y., Costanzo, S., Davis, R.E. 2019. Rapid detection and identification of ‘Candidatus Phytoplasma pini’-related strains in Lithuania based on genomic markers present in 16S rRNA and tuf genes. Forest Pathology. 49:e12553. https://doi.org/10.1111/efp.12553.
DOI: https://doi.org/10.1111/efp.12553

Interpretive Summary: Phytoplasmas are small bacteria that reside in plant nutrient-conducting veins and are spread by insects that feed on infected plants. These bacteria are responsible for numerous diseases in agriculturally and environmentally important plants globally. Since phytoplasmas cannot be cultured under aseptic conditions in laboratory, they are identified based on molecular characteristics. 'Candidatus Phytoplasma pini' is a phytoplasma that seriously damage pine trees and was first discovered in Europe. In this work, ARS scientists and collaborators in Lithuania analyzed DNA from a phytoplasma that infects pine trees growing in forests in Lithuania. The research team determined the nucleotide sequences of a cluster of essential genes and identified unique nucleotide sequence blocks that can be used as molecular markers for specific detection and identification of the pine phytoplasma. Findings from the work expands knowledge of distinct genomic features of the pine phytoplasma. The findings are important to scientists and extension personnel who are concerned with phytoplasmal disease diagnosis and management. The information is also critical to regulatory agencies for preventing exotic pathogens from being introduced into the U.S.

Technical Abstract: In order to devise a method for rapid detection of 'Candidatus (Ca.) Phytoplasma pini' and for distinguishing it rapidly from other phytoplasmas, we carried out preliminary sequencing of Lithuanian 'Ca. Phytoplasma pini' strain PineBL2 using Illumina (NGS) technology. We focused on a resulting 6,358 bp contig of strain PineBL2 containing a partial DNA-dependent RNA polymerase (DpRp) subunit Rpo' gene (rpoC), a ribosomal protein Rps12 gene (rpsL), a ribosomal protein Rps7 gene (rpsG), an elongation factor EF-G gene (fusA), a translation elongation factor EF-TU gene (tuf), and a gene encoding a hypothetical protein (a DAK2 domain fusion protein YloV). Based on alignments of this region with that of other phytoplasmas, we designed new tuf gene primer pair for PCR-based detection of 'Ca. Phytoplasma pini'. Because 'Ca. Phytoplasma pini' strains are expected to reside in the pine phloem in a very low titre, one might expect that they could be detected only by nested PCR. By contrast, the primers and protocols designed for direct PCR in the current work made possible direct PCRs for fast detection and identification of 'Ca. Phytoplasma pini' by amplifying 484 bp 16S rDNA and 513 bp tuf gene fragments containing regions unique to this phytoplasma.